Two components of the primitive respiratory chain of Vitreoscilla have been purified and characterized in this laboratory, cytochrome o, the terminal oxidase, and a non-heme iron flavoprotein reductase. Two intermediates in the reaction of cytochrome o with oxygen have been identified both in vitro and in respiring whole cells: compound D, a dimer, (b2b'2)202, and the oxygenated compound, b2b'2-02. Experiments are described to test directly the model proposed for the reaction of cytochrome o with oxygen. Specific chemical properties of compound D will be examined as well as further reaction of the cytochrome with oxygen, ligands, and hydrogen peroxide, formation of other intermediates, their temperature stability, and rates of turnover. Techniques for these studies will include protein crosslinking, visible and infrared spectroscopy, stopped-flow, and low temperature titrations. We will look for oxygenated intermediates in whole cells using low temperature techniques. Cells with heme content experimentally varied will be used to correlate cytochrome content and respiratory activity, e. g., level of oxygenated intermediates in the respiring steady state. The protein effector of the NADH-cytochrome o reductase system will be purified and characterized to study its role and the role of the flavoprotein reductase in the reaction with cytochrome o and to enable reconstitution of the NADH-cytochrome o oxidase system with high activity. Respiratory membrane fragments will be used to test correspondence of results obtained with the purified solubilized components with their role in vivo, such as generation of hydrogen peroxide by the NADH-cytochrome o oxidase system. These membrane fragments are capable of energy conservation during NADH oxidation and an attempt will be made to obtain a soluble bacterial oxidative phosphorylating system with cytochrome o as the terminal oxidase. Cyanide and CO resistant mutants of Vitreoscilla have been isolated and the respiration and cytochrome of these mutants will be characterized.